The present invention relates to substances which bind with high affinity to endotoxin (lipopolysaccharide [LPS]), and which are useful for the prevention or treatment of a variety of conditions and diseases, such as of Gram-negative and Gram-positive bacterial sepsis, or bacterial or fungal infections. Furthermore, said substances may be used for neutralizing effects associated with heparin. The substances are LPS-binding peptides comprising an LPS-binding domain. The invention also encompasses methods for the detection and removal of bacteria LPS from solutions.
In humans, LPS released during infection by Gram-negative bacteria can cause the severe pathological changes associated with septic shock (Duma, Am. J. Med. 78 (1985) 154-163; Glauser et al., Lancet 338 (1991), 732-736). In the United States, septic shock is responsible for between 100,000 and 300,000 deaths annually (Ziegler et al., N. Eng. J. Med. 324 (1991), 429-436) and in Germany for between 70,000 and 100,000. Although a variety of agents have been evaluated for neutralizing LPS or enhancing its clearance in vivo, there remains no specific treatment for Gram-negative bacterial sepsis.
LPS is a glycolipid that is ubiquitous in the outer membrane of Gram-negative bacteria (Raetz, Annu. Rev. Biochem. 59 (1990), 129-170). LPS consists of an oligosaccharide and a lipid portion and is characterized by an overall negative charge, stability to heat, and high molecular weight. While the chemical structure of most LPS molecules is complex and diverse, a common feature is the lipid A region. Lipid A, the membrane anchor of LPS, consists of a central phosphodisaccharide unit that is attached to up to seven fatty acid chains. Most of the biological activities of LPS reside in the lipid A portion (Galanos et al., Eur. J. Biochem. 145, (1985), 1-5).
Septic shock is complex condition which arises from a cascade of molecular and cellular events following infection by microorganisms, predominant among which are Gram-negative bacteria. The onset of shock arises from the interaction of LPS or lipid A with membrane-bound receptors on macrophages and blood monocytes (Couturier et al., J. Immun. 147 (1991), 1899-1904) or various serum proteins, such as the septins (Wright et al., J. Exp. Med. 176 (1992), 719-727). These interactions lead to an increase in the levels of pro-inflammatory mediators such as tumor necrosis factor, IL-1, IL-6, and interferon-c. Endothelial cells are also stimulated to produce factors which attract neutrophils. Release of enzymes and other factors by activated neutrophils causes damage to local vasculature which can lead rapidly to death.
One approach to the treatment of sepsis is the use of substances which bind to LPS and neutralize its toxic effects in vivo. Although there are numerous proteins which bind LPS, the number of substances which effectively neutralize LPS in vivo are very few. A number of such substances have been identified, including polymyxins (Morrison et al., Immunochem. 13 (1976), 813-818), polymyxin-derived peptides (Rustici et al., Science 259 (1993), 361-365) , polyclonal (Ziegler et al., N. Eng. J. Med. 307 (1982), 1225-1230) and monoclonal (Ziegler et al. (1991), loc. cit.) antibodies, bactericidal/permeability-increasing protein (BPI) (Marra et al., J. Immun. 148 (1992), 532-537), lipopolysaccharide binding protein (LBP) (Schuhmann et al., Science 249 (1990), 1429-1431), and Limulus anti-LPS factor (LALF) (Akategawa et al., J. Biol. Chem. 261 (1986), 7357-7365, Muta et al., J. Biochem. 101 (1987), 1321-1330).
The simplest molecules that bind to the lipid A portion of LPS with high affinity are the polymyxin antibiotics; these are positively charged amphipathic cyclic oligopeptides attached to a lipid tail. Although polymyxins bind to LPS/lipid A with high affinity, they suffer the drawback from a therapeutic stand-point of having unacceptably high toxicity (Craig et al., Infect. Immun. 10 (1974), 287-292). The LPS-binding monoclonal antibodies HA-1A and E5 both failed to demonstrate positive clinical effects for the treatment of Gram-negative septic shock. One of the main problems associated with these antibodies is non-specificity; for example, HA-1A binds tightly to numerous hydrophobic structures apart from lipid A (see, for example, Baumgartner et al., J. Exp. Med. 171 (1990), 889-896). The human proteins BPI and LBP are both being investigated for the treatment of Gram-negative sepsis (Marra et al., loc. cit.; Ulevitch et al. (1986), WP 86/06279). BPI, which s stored in specific granules of polymorphonuclear cells, kills Gram-negative bacteria by binding to membrane-bound LPS and disrupting the permeability barrier. LBP is a mammalian serum protein which also binds very tightly to LPS. Although LBP shares sequence homology with BPI (Schuhmann et al., loc. cit.), it is not directly cytotoxic to Gram-negative bacteria and its precise function is obscure. Most recently, LALF has been investigated for use in sepsis (Warren et al., Infect. Immun. 60 (1992), 2506-2513; Wainwright et al. (1992) WO 92/20715). This protein is almost certain to suffer the disadvantages associated with other foreign proteins for human therapy; it is immunogenic and has only a short half-life in circulation. These factors will reduce its clinical potential. None of these substances have been proven to be effective for the treatment of the serious conditions associated with Gram-negative infection.
Thus, the technical problem underlying the present invention is to provide substances which bind LPS released by Gram-negative bacteria, neutralize its toxic effects, and exhibit no toxicity.
The solution to the above technical problem was achieved by providing substances which relate to peptides which bind tightly to LPS, and therefore have utility in the diagnosis and treatment of Gram-negative and other septic conditions.
Thus, the present invention relates to LPS-binding peptides comprising an LPS-binding domain comprising at least:
(a) the amino acid sequence 1-2-3-4-5-6-7-8, wherein the numbers represent any of the following amino acids:
1=a polar or positively charged amino acid, preferably C, H, K, N, Q, R, S, T, W, or Y;
2=a hydrophobic amino acid, preferably A, F, H, I, L, M, V, or W;
3=a basic amino acid, preferably H, K, or R;
4=a hydrophobic or positively charged amino acid, preferably A, F, H, I, K, L, M, R, V, or W;
5=a hydrophobic, polar, or positively charged amino acid, preferably A, C, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;
6=a positively charged amino acid, preferably K or R;
7=A hydrophobic, polar, or positively charged amino acid, preferably A, C, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;
8=a hydrophobic or positively charged amino acid, preferably A, F, H, I, K, L, M, R, V, or W;
(b) a corresponding inverse amino acid sequence; or
(c) a variation of said amino acid sequence (a) or (b) capable of effectively binding to LPS.
The peptides of the present invention effectively bind to LPS, i.e. they interact specifically with LPS with an association constant greater than 105 Mxe2x88x921. In this context, an LPS-binding peptide is a chain of amino acids linked to each other by peptide bonds. An LPS-binding domain is the shortest possible chain of amino acids within an LPS-binding peptide which effectively binds to LPS.
All peptide structures disclosed use the single letter code for amino acids.